A new body of evidence is emerging which indicates that ethanol can interact with highly specific sites on specialized proteins to modulate their functional activities. Ethanol was found to activate nicotinic acetylcholine, GABAA and serotonin receptors, and to inhibit NMDA receptors and adenosine uptake via the nucleoside transporter. Moreover, in at least two cases, the interaction of ethanol with these sites appears to require phosphorylation of the proteins. An eight amino acid sequence in gamma subunit of the GABAA receptor that confers sensitivity to ethanol contains a consensus PKC phosphorylation site. Moreover, inhibition of the adenosine transporter by ethanol requires the presence of cAMP-dependent protein kinase; no inhibition occurs in mutants lacking this enzyme. The inhibition of nucleoside transport by ethanol is specific for only one subtype of a family of nucleoside transporters. This inhibition of nucleoside transport by ethanol is specific for only one subtype of a family of nucleoside transporters. this inhibition is important for the acute and chronic effects of ethanol on the cAMP signal transduction system which may be responsible for many of the pleiotropic effects of chronic alcoholic consumption. We have found that inhibition of adenosine uptake leads to an extracellular accumulation of adenosine which in turn is responsible for the heterologous desensitization that occurs in many cultured cell lines and freshly isolated lymphocytes from alcoholics. In this proposal, we describe our plans to elucidate the molecular basis for the sensitivity of this facilitative nucleoside transporter to ethanol. To this end, we plan to clone a family of facilitative nucleoside transporters. We will also identify which domain(s) of the transporter molecule confers sensitivity to ethanol and whether phosphorylation of the transporter regulates ethanol sensitivity. The specific aims of this study are: (1) to clone the NBMPR-sensitive nucleoside transporter by expression cloning in Xenopus oocytes or by PCR-based homology cloning (2) to determine the relationship between phosphorylation of the transporter and its sensitivity to ethanol by constructing chimeric fusion proteins and by site-directed mutagenesis of the nucleoside transporter (3) to determine how phosphorylation of the transporter regulates ethanol sensitivity in human erythrocytes. These studies identifying the molecular mechanisms responsible for regulating ethanol-sensitivity of the nucleoside transporter may lead to the development of unique tools to obtain specific markers of alcoholism, develop new probes for investigating genetic alcoholism, and explore new therapies to prevent or reverse alcohol dependence.